Effects of follicle-stimulating hormone on energy balance and tissue metabolic health after loss of ovarian function.

Loss of ovarian function imparts increased susceptibility to obesity and metabolic disease. These effects are largely attributed to decreased estradiol (E2), but the role of increased follicle-stimulating hormone (FSH) in modulating energy balance has not been fully investigated. Previous work that blocked FSH binding to its receptor in mice suggested this hormone may play a part in modulating body weight and energy expenditure after ovariectomy (OVX). We used an alternate approach to isolate the individual and combined contributions of FSH and E2 in mediating energy imbalance and changes in tissue-level metabolic health. Female Wistar rats were ovariectomized and given the gonadotropin releasing hormone (GnRH) antagonist degarelix to suppress FSH production. E2 and FSH were then added back individually and in combination for a period of 3 wk. Energy balance, body mass composition, and transcriptomic profiles of individual tissues were obtained. In contrast to previous studies, suppression and replacement of FSH in our paradigm had no effect on body weight, body composition, food intake, or energy expenditure. We did, however, observe organ-specific effects of FSH that produced unique transcriptomic signatures of FSH in retroperitoneal white adipose tissue. These included reductions in biological processes related to lipogenesis and carbohydrate transport. In addition, rats administered FSH had reduced liver triglyceride concentration (P < 0.001), which correlated with FSH-induced changes at the transcriptomic level. Although not appearing to modulate energy balance after loss of ovarian function in rats, FSH may still impart tissue-specific effects in the liver and white adipose tissue that might affect the metabolic health of those organs.NEW & NOTEWORTHY We find no effect of follicle-stimulating hormone (FSH) on energy balance using a novel model in which rats are ovariectomized, subjected to gonadotropin-releasing hormone antagonism, and systematically given back FSH by osmotic pump. However, tissue-specific effects of FSH on adipose tissue and liver were observed in this study. These include unique transcriptomic signatures induced by the hormone and a stark reduction in hepatic triglyceride accumulation.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

Inactivation of Tsc2 in Abcg2 lineage-derived cells drives the appearance of polycystic lesions and fibrosis in the adult kidney.

Tuberous sclerosis complex 2 (TSC2), or tuberin, is a pivotal regulator of the mechanistic target of rapamycin signaling pathway that controls cell survival, proliferation, growth, and migration. Loss of Tsc2 function manifests in organ-specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of the kidney has identified ATP-binding cassette G2 (Abcg2) expression in renal proximal tubules of adult mice as well as a in a novel cell population. The impact in adult kidney of Tsc2 knockdown in the Abcg2-expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36-37 with an intact 3' region and polycystin 1, is driven by Abcg2. Here, we demonstrate that selective expression of Tsc2fl36-37 in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells, which results in progressive proximal tubule injury, impaired kidney function, formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2-expressing proximal tubule epithelium and mesenchyme during the development of cystic lesions and remodeling of kidney parenchyma.

Modulation of Energy Expenditure by Estrogens and Exercise in Women.

Reducing estrogen in women results in decreases in energy expenditure, but the mechanism(s) remain largely unknown. We postulate that the loss of estrogens in women is associated with increased accumulation of bone marrow-derived adipocytes in white adipose tissue, decreased activity of brown adipose tissue, and reduced levels of physical activity. Regular exercise may counteract the effects of estrogen deficiency.

De novo generation of adipocytes from circulating progenitor cells in mouse and human adipose tissue.

White adipocytes in adults are typically derived from tissue resident mesenchymal progenitors. The recent identification of de novo production of adipocytes from bone marrow progenitor-derived cells in mice challenges this paradigm and indicates an alternative lineage specification that adipocytes exist. We hypothesized that alternative lineage specification of white adipocytes is also present in human adipose tissue. Bone marrow from transgenic mice in which luciferase expression is governed by the adipocyte-restricted adiponectin gene promoter was adoptively transferred to wild-type recipient mice. Light emission was quantitated in recipients by in vivo imaging and direct enzyme assay. Adipocytes were also obtained from human recipients of hematopoietic stem cell transplantation. DNA was isolated, and microsatellite polymorphisms were exploited to quantify donor/recipient chimerism. Luciferase emission was detected from major fat depots of transplanted mice. No light emission was observed from intestines, liver, or lungs. Up to 35% of adipocytes in humans were generated from donor marrow cells in the absence of cell fusion. Nontransplanted mice and stromal-vascular fraction samples were used as negative and positive controls for the mouse and human experiments, respectively. This study provides evidence for a nontissue resident origin of an adipocyte subpopulation in both mice and humans.

Saxagliptin restores vascular mitochondrial exercise response in the Goto-Kakizaki rat.

Cardiovascular disease risk and all-cause mortality are largely predicted by physical fitness. Exercise stimulates vascular mitochondrial biogenesis through endothelial nitric oxide synthase (eNOS), sirtuins, and PPARγ coactivator 1α (PGC-1α), a response absent in diabetes and hypertension. We hypothesized that an agent regulating eNOS in the context of diabetes could reconstitute exercise-mediated signaling to mitochondrial biogenesis. Glucagon-like peptide 1 (GLP-1) stimulates eNOS and blood flow; we used saxagliptin, an inhibitor of GLP-1 degradation, to test whether vascular mitochondrial adaptation to exercise in diabetes could be restored. Goto-Kakizaki (GK) rats, a nonobese, type 2 diabetes model, and Wistar controls were exposed to an 8-day exercise intervention with or without saxagliptin (10 mg·kg·d). We evaluated the impact of exercise and saxagliptin on mitochondrial proteins and signaling pathways in aorta. Mitochondrial protein expression increased with exercise in the Wistar aorta and decreased or remained unchanged in the GK animals. GK rats treated with saxagliptin plus exercise showed increased expression of mitochondrial complexes, cytochrome c, eNOS, nNOS, PGC-1α, and UCP3 proteins. Notably, a 3-week saxagliptin plus exercise intervention significantly increased running time in the GK rats. These data suggest that saxagliptin restores vascular mitochondrial adaptation to exercise in a diabetic rodent model and may augment the impact of exercise on the vasculature.

ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling.

Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-ß1 (TGF-ß1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-ß1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-ß1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

Identification of a common Wnt-associated genetic signature across multiple cell types in pulmonary arterial hypertension.

Understanding differences in gene expression that increase risk for pulmonary arterial hypertension (PAH) is essential to understanding the molecular basis for disease. Previous studies on patient samples were limited by end-stage disease effects or by use of nonadherent cells, which are not ideal to model vascular cells in vivo. These studies addressed the hypothesis that pathological processes associated with PAH may be identified via a genetic signature common across multiple cell types. Expression array experiments were initially conducted to analyze cell types at different stages of vascular differentiation (mesenchymal stromal and endothelial) derived from PAH patient-specific induced pluripotent stem (iPS) cells. Molecular pathways that were altered in the PAH cell lines were then compared with those in fibroblasts from 21 patients, including those with idiopathic and heritable PAH. Wnt was identified as a target pathway and was validated in vitro using primary patient mesenchymal and endothelial cells. Taken together, our data suggest that the molecular lesions that cause PAH are present in all cell types evaluated, regardless of origin, and that stimulation of the Wnt signaling pathway was a common molecular defect in both heritable and idiopathic PAH.

Targeting mitochondria to restore failed adaptation to exercise in diabetes.

Our translational research group focuses on addressing the problem of exercise defects in diabetes with basic research efforts in cell and rodent models and clinical research efforts in subjects with diabetes mellitus. CREB (cAMP-response-element-binding protein) regulates cellular differentiation of neurons, ß-cells, adipocytes and smooth muscle cells; it is also a potent survival factor and an upstream regulator of mitochondrial biogenesis. In diabetes and cardiovascular disease, CREB protein content is decreased in the vascular media, and its regulation in aberrant in ß-cells, neurons and cardiomyocytes. Loss of CREB content and function leads to decreased vascular target tissue resilience when exposed to stressors such as metabolic, oxidative or sheer stress. This basic research programme set the stage for our central hypothesis that diabetes-mediated CREB dysfunction predisposes the diabetes disease progression and cardiovascular complications. Our clinical research programme revealed that diabetes mellitus leads to defects in functional exercise capacity. Our group has determined that the defects in exercise correlate with insulin resistance, endothelial dysfunction, decreased cardiac perfusion and diastolic dysfunction, slowed muscle perfusion kinetics, decreased muscle perfusion and slowed oxidative phosphorylation. Combined basic and clinical research has defined the relationship between exercise and vascular function with particular emphasis on how the signalling to CREB and eNOS [endothelial NOS (nitric oxide synthase)] regulates tissue perfusion, mitochondrial dynamics, vascular function and exercise capacity. The present review summarizes our current working hypothesis that restoration of eNOS/NOS dysfunction will restore cellular homoeostasis and permit an optimal tissue response to an exercise training intervention.

Inhibition of phosphatidylinositol 3-kinase/Akt signaling attenuates hypoxia-induced pulmonary artery remodeling and suppresses CREB depletion in arterial smooth muscle cells.

Hypoxia-induced pulmonary hypertension is characterized by progressive remodeling of the pulmonary artery (PA) system and loss of the transcription factor, cAMP response element binding protein (CREB) in PA smooth muscle cells (SMCs). Previous in vitro studies suggested that platelet-derived growth factor, a mitogen produced in the hypoxic arterial wall, elicits loss of CREB in medial SMCs via the PI3K/Akt pathway. These events trigger switching of SMCs from a quiescent, contractile phenotype to a proliferative, migratory, dedifferentiated, and synthetic phenotype, which contributes to PA thickening. Here, we investigated whether inhibition of PI3K or Akt could attenuate arterial remodeling in the lung and prevent CREB loss in PA medial SMCs in rats subjected to chronic hypoxia. Inhibition of either enzyme-blunted hypoxia-induced PA remodeling and SMC CREB depletion and diminished SMC proliferation and collagen deposition. Inhibition of Akt, but not PI3K, suppressed muscularization of distal arterioles and blunted right ventricular hypertrophy. Interestingly, mean PA pressure was elevated equally by hypoxia in untreated and inhibitor-treated groups but was normalized acutely by the Rho kinase inhibitor, Fasudil. We conclude that PI3K and Akt inhibitors can attenuate hypoxia-induced PA remodeling and SMC CREB depletion but fail to block the development of pulmonary hypertension because of their inability to repress Rho kinase-mediated vasoconstriction.

De novo generation of white adipocytes from the myeloid lineage via mesenchymal intermediates is age, adipose depot, and gender specific.

It is generally assumed that white adipocytes arise from resident adipose tissue mesenchymal progenitor cells. We challenge this paradigm by defining a hematopoietic origin for both the de novo development of a subset of white adipocytes in adults and a previously uncharacterized adipose tissue resident mesenchymal progenitor population. Lineage and cytogenetic analysis revealed that bone marrow progenitor (BMP)-derived adipocytes and adipocyte progenitors arise from hematopoietic cells via the myeloid lineage in the absence of cell fusion. Global gene expression analysis indicated that the BMP-derived fat cells are bona fide adipocytes but differ from conventional white or brown adipocytes in decreased expression of genes involved in mitochondrial biogenesis and lipid oxidation, and increased inflammatory gene expression. The BMP-derived adipocytes accumulate with age, occur in higher numbers in visceral than in subcutaneous fat, and in female versus male mice. BMP-derived adipocytes may, therefore, account in part for adipose depot heterogeneity and detrimental changes in adipose metabolism and inflammation with aging and adiposity.

Concise review: adipocyte origins: weighing the possibilities.

Adipose tissue is the primary energy reservoir in the body and an important endocrine organ that plays roles in energy homeostasis, feeding, insulin sensitivity, and inflammation. While it was tacitly assumed that fat in different anatomical locations had a common origin and homogenous function, it is now clear that regional differences exist in adipose tissue characteristics and function. This is exemplified by the link between increased deep abdominal or visceral fat, but not peripheral adipose tissue and the metabolic disturbances associated with obesity. Regional differences in fat function are due in large part to distinct adipocyte populations that comprise the different fat depots. Evidence accrued primarily in the last decade indicates that the distinct adipocyte populations are generated by a number of processes during and after development. These include the production of adipocytes from different germ cell layers, the formation of distinct preadipocyte populations from mesenchymal progenitors of mesodermal origin, and the production of adipocytes from hematopoietic stem cells from the bone marrow. This review will examine each of these process and their relevance to normal adipose tissue formation and contribution to obesity-related diseases.

Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo.

Reprogramming somatic cells into an ESC-like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8­12 weeks. Differentiation was assessed by lineage-specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS-derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage-specific genes. Isolated implants of iPS cell-derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease.

Hematopoietic Stem Cell-Derived Adipocytes Modulate Adipose Tissue Cellularity, Leptin Production and Insulin Responsiveness in Female Mice.

A subpopulation of adipocytes in the major adipose depots of mice is produced from hematopoietic stem cells rather than mesenchymal progenitors that are the source of conventional white and brown/beige adipocytes. To analyze the impact of hematopoietic stem cell-derived adipocytes (HSCDAs) in the adipose niche we transplanted HSCs in which expression of a diphtheria toxin gene was under the control of the adipocyte-specific adiponectin gene promoter into irradiated wild type recipients. Thus, only adipocytes produced from HSC would be ablated while conventional white and brown adipocytes produced from mesenchymal progenitor cells would be spared. Wild type mice transplanted with HSCs from mice containing a reporter gene, but not the diphtheria toxin gene, regulated by the adiponectin gene promoter served as controls. In mice in which HSCDA production was suppressed, adipocyte size declined while adipose depot weights were unchanged and the number of conventional adipocyte progenitors significantly increased. We also measured a paradoxical increase in circulating leptin levels while physical activity was significantly decreased in the HSCDA depleted mice. Finally, insulin sensitivity was significantly reduced in HSCDA depleted mice. In contrast, loss of HSCDA production had no effect on body weight, components of energy balance, or levels of several circulating adipokines and tissue-resident inflammatory cells. These data indicate that ablation of this low-abundance subpopulation of adipocytes is associated with changes in circulating leptin levels and leptin-regulated endpoints associated with adipose tissue function. How they do so remains a mystery, but our results highlight the need for additional studies to explore the role of HSCDAs in other physiologic contexts such as obesity, metabolic dysfunction or loss of sex hormone production.

CREB downregulation in vascular disease: a common response to cardiovascular risk.

OBJECTIVE: To examine the impact of low-density lipoprotein (LDL), an established mediator of atherosclerosis, on the transcription factor cAMP-response element-binding protein (CREB), which is a regulator of vascular smooth muscle cell (VSMC) quiescence. METHODS AND RESULTS: VSMC CREB content is diminished in rodent models of diabetes and pulmonary hypertension. We examined aortic CREB content in rodent models of aging, hypertension, and insulin resistance, and we determined nuclear CREB protein in the medial VSMC of high-fat-fed LDL receptor-null mice. There was significant loss of CREB protein in all models. In vitro, primary culture rat aortic VSMC exposed to LDL and oxidized LDL exhibited a rapid, transient increase in CREB phosphorylation and transient phosphorylation/activation of Akt, ERK, JNK, ans p38 MAPK. Exposure to oxidized LDL, but not to LDL, for 24 to 48 hours decreased CREB protein in a dose-dependent fashion and led to nuclear exclusion of CREB. Pharmacological reactive oxygen species scavengers and inhibition of ERK activation blocked oxidized LDL-mediated CREB downregulation. CONCLUSIONS: These data support a model wherein loss of VSMC CREB protein, which renders these cells more susceptible to activation and apoptosis, is a common pathological response to vascular injury and potentially contributes to plaque progression.

Reduction of reactive oxygen species prevents hypoxia-induced CREB depletion in pulmonary artery smooth muscle cells.

Hypoxia-induced pulmonary arterial hypertension (PAH) is a deadly disease characterized by progressive remodeling and persistent vasoconstriction of the pulmonary arterial system. Remodeling of the pulmonary artery (PA) involves smooth muscle cell (SMC) proliferation, hypertrophy, migration, and elevated extracellular matrix (ECM) production elicited by mitogens and oxidants produced in response to hypoxic insult. We previously reported that the transcription factor cAMP response element binding protein (CREB) is depleted in medial PA SMCs in remodeled, hypertensive vessels in rats or calves exposed to chronic hypoxia. In culture, CREB loss can be induced in PA SMCs by exogenous oxidants or platelet-derived growth factor. Forced depletion of CREB with small interfering RNA (siRNA) in PA SMCs is sufficient to induce their proliferation, hypertrophy, migration, dedifferentiation, and ECM production. This suggests that oxidant and/or mitogen-induced loss of CREB in medial SMCs is, in part, responsible for PA thickening. Here, we tested whether oxidant scavengers could prevent the loss of CREB in PA SMCs and inhibit SMC proliferation, migration, and ECM production using in vitro and in vivo models. Exposure of PA SMCs to hypoxia induced hydrogen peroxide (H2O2) production and loss of CREB. Treatment of SMCs with exogenous H2O2 or a second oxidant, Sin-1, elicited CREB depletion under normoxic conditions. Exogenous H2O2 also induced SMC proliferation, migration, and increased elastin levels as did forced depletion of CREB. In vivo, hypoxia-induced thickening of the PA wall was suppressed by the superoxide dismutase mimetic, Tempol, which also prevented the loss of CREB in medial SMCs. Tempol also reduced hypoxia-induced SMC proliferation and elastin deposition in the PA. The data indicate that CREB levels in the arterial wall are regulated in part by oxidants produced in response to hypoxia and that CREB plays a crucial role in regulating SMC phenotype and PA remodeling.

Metabolite G-Protein Coupled Receptors in Cardio-Metabolic Diseases.

G protein-coupled receptors (GPCRs) have originally been described as a family of receptors activated by hormones, neurotransmitters, and other mediators. However, in recent years GPCRs have shown to bind endogenous metabolites, which serve functions other than as signaling mediators. These receptors respond to fatty acids, mono- and disaccharides, amino acids, or various intermediates and products of metabolism, including ketone bodies, lactate, succinate, or bile acids. Given that many of these metabolic processes are dysregulated under pathological conditions, including diabetes, dyslipidemia, and obesity, receptors of endogenous metabolites have also been recognized as potential drug targets to prevent and/or treat metabolic and cardiovascular diseases. This review describes G protein-coupled receptors activated by endogenous metabolites and summarizes their physiological, pathophysiological, and potential pharmacological roles.

Hematopoietic stem cells produce intermediate lineage adipocyte progenitors that simultaneously express both myeloid and mesenchymal lineage markers in adipose tissue.

Some adipocytes are produced from bone marrow hematopoietic stem cells. In vitro studies previously indicated that these bone marrow-derived adipocytes (BMDAs) were generated from adipose tissue macrophage (ATM) that lose their hematopoietic markers and acquire mesenchymal markers prior to terminal adipogenic differentiation. Here we interrogated whether this hematopoietic-to-mesenchymal transition drives BMDA production In vitro. We generated transgenic mice in which the lysozyme gene promoter (LysM) indelibly labeled ATM with green fluorescent protein (GFP). We discovered that adipose stroma contained a population of LysM-positive myeloid cells that simultaneously expressed hematopoietic/myeloid markers (CD45 and CD11b), and mesenchymal markers (CD29, PDGFRa and Sca-1) typically found on conventional adipocyte progenitors. These cells were capable of adipogenic differentiation In vitro and In vitro, while other stromal populations deficient in PDGFRa and Sca-1 were non-adipogenic. BMDAs and conventional adipocytes expressed common fat cell markers but exhibited little or no expression of hematopoietic and mesenchymal progenitor cell markers. The data indicate that BMDAs are produced from ATM simultaneously expressing hematopoietic and mesenchymal markers rather than via a stepwise hematopoietic-to-mesenchymal transition. Because BMDA production is stimulated by high fat feeding, their production from hematopoietic progenitors may maintain adipocyte production when conventional adipocyte precursors are diminished.

Neprilysin null mice develop exaggerated pulmonary vascular remodeling in response to chronic hypoxia.

Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder.

Thiazolidinediones prevent PDGF-BB-induced CREB depletion in pulmonary artery smooth muscle cells by preventing upregulation of casein kinase 2 alpha' catalytic subunit.

BACKGROUND: The transcription factor CREB is diminished in smooth muscle cells (SMCs) in remodeled, hypertensive pulmonary arteries (PAs) in animals exposed to chronic hypoxia. Forced depletion of cyclic adenosine monophosphate response element binding protein (CREB) in PA SMCs stimulates their proliferation and migration in vitro. Platelet-derived growth factor (PDGF) produced in the hypoxic PA wall promotes CREB proteasomal degradation in SMCs via phosphatidylinositol-3-kinase/Akt signaling, which promotes phosphorylation of CREB at 2 casein kinase 2 (CK2) sites. Here we tested whether thiazolidinediones, agents that inhibit hypoxia-induced PA remodeling, attenuate SMC CREB loss. METHODS: Depletion of CREB and changes in casein kinase 2 catalytic subunit expression and activity were measured in PA SMC treated with PDGF. PA remodeling and changes in medial PA CREB and casein kinase 2 levels were evaluated in lung sections from rats exposed to hypoxia for 21 days. RESULTS: We found that the thiazolidinedione rosiglitazone prevented PA remodeling and SMC CREB loss in rats exposed to chronic hypoxia. Likewise, the thiazolidinedione troglitazone blocked PA SMC proliferation and CREB depletion induced by PDGF in vitro. Thiazolidinediones did not repress Akt activation by hypoxia in vivo or by PDGF in vitro. However, PDGF-induced CK2 alpha' catalytic subunit expression and activity in PA SMCs, and depletion of CK2 alpha' subunit prevented PDGF-stimulated CREB loss. Troglitazone inhibited PDGF-induced CK2 alpha' subunit expression in vitro and rosiglitazone blocked induction of CK2 catalytic subunit expression by hypoxia in PA SMCs in vivo. CONCLUSION: We conclude that thiazolidinediones prevent PA remodeling in part by suppressing upregulation of CK2 and loss of CREB in PA SMCs.